Rituximab (RTX) and Ofatumumab (OFA) efficacies in cancer therapy depend in part on the induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of membrane attack complex (MAC), thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell malignancies such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) and up-regulation of hCD59 expression is an important determinant of the sensitivity of those cancer cells to RTX treatment. Therefore, it is imperative for us to develop a molecule capable of abrogating CD59 function in cancer cells and thus facilitate antibody- mediated cancer therapy. However, targeted toxicity effect from antibody specific against hCD59 and less efficacy of C8 or C9 peptides limit them for therapeutic purposes. Recently, we developed a specific and potent hCD59 inhibitor, rILYd4 (114aa), and demonstrated that rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing RTX resistant lymphoma cells and primary CLL to RTX and OFA treatment. By defining PK/PD profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. We demonstrated that increasing expression level of hCD59 in RTX-resistant cells occurs due to selection of pre-existing clones, rather than de novo induction of hCD59. We also demonstrated that the sensitivity to CDC effects mediated by OFA or RTX on RTX-resistant B-cell malignant cell lines and CLL cells negatively correlated with the level of CD59 on the cell surface. Taken together, our results rationalize the use of rILYd4 as a therapeutic adjuvant for RTX and OFA treatment of RTX-resistant NHL and CLL. Based on these results, we propose to optimize rILYd4 and develop less or non immunogenic, potent and stable rILYd4-derived peptide. We hypothesize that the optimized rILYd4 will improve the physicochemical properties of parental rILYd4, eliminate or reduce markedly immunogenicity, increase metabolic stability and half life in circulation, and enhance therapeutic efficacy, thereb leading to effective rILYd4-related therapeutics for enhancing as a adjuvant to anti-cancer drugs such as OFA and RTX in B-cell malignancies that have relapsed after prior antibody-based therapies. We propose to 1) to identify the sequences in rILYd4 responsible for its anti-hCD59 activity by phage display and synthetic approaches, and use them to develop a small, potent and non-immunogenic rILYd4-derived peptide (derivative) (Aim 1) and 2) to study its immune responses in human HLA transgenic mice and characterize its PK/PD, efficacy and toxicity profiles in humanized hCD59 transgenic mice. (Aim 2). Successful outcome of this research will generate a novel therapeutic rILYd4-derived agent for clinical trials that will test its efficacy a an adjuvant for RTX- or OFA-based B-cell malignancy therapy and provide the much needed means to overcome the devastating RTX- or OFA-resistant B-cell malignancies.